液滴微流控筛选提高毕赤酵母异源蛋白分泌量
Improving the secretion of heterologous proteins in Pichia pastoris based on droplet microfluidic screening
巴斯德毕赤酵母是工业发酵中异源蛋白生产的重要宿主,但毕赤酵母表达系统的潜力尚未得到充分的探索。为了提高毕赤酵母外源蛋白的分泌能力,通过常压室温等离子体诱变(ARTP)构建毕赤酵母突变体文库,使用高通量液滴微流控技术对文库进行筛选。经过四轮迭代诱变与分选,得到了4株蛋白质分泌量提高的突变体,可提高模式蛋白α-淀粉酶相对酶活150%,提高人血清白蛋白(HSA)的分泌量110%,提高木聚糖酶酶活60%。对高分泌菌株进行全基因组重测序,鉴定出31个潜在的促进毕赤酵母蛋白质分泌的突变位点,这些突变位点绝大部分尚无文献详细报道研究过。该研究中分选出的突变体可作为发酵工业中高产异源蛋白的平台菌株,发现的突变基因可以用于反向代谢工程,为构建高效的蛋白质分泌细胞工厂提供研究方向。
Pichia pastoris serves as a crucial host for the industrial production of heterologous protein. However, the potential of Pichia expression system has not been fully explored. In order to enhance the secretion ability of exogenous proteins in Pichia pastoris, a mutant library, generated through atmospheric pressure room temperature plasma mutagenesis (ARTP), were systematically screened using high-throughput droplet microfluidic technology. After four rounds of iterative mutagenesis and sorting, four mutant strains with enhanced protein secretion were obtained, which demonstrated remarkable 150% increase in the relative enzyme activity of pattern protein α-amylase. Moreover, the secretion proficiency for human serum albumin (HSA) in these mutants surpassed that of the original strain by 110%, xylanase activity increased by 60%. Whole genome resequencing analysis of high secretion strains revealed 31 mutations potentially related to increased protein secretion. Notably, most of these mutation sites have not been extensively reported or investigated in previous literature. The mutants selected in this study can serve as promising platform strains for high-yield production of heterologous proteins. The identified mutated genes offer valuable targets for reverse metabolic engineering, proving the way for constructing efficient protein secretion cell factories and providing crucial insights for future research directions.
郑梦林、杨艳坤、白仲虎
生物工程学遗传学分子生物学
毕赤酵母液滴微流控蛋白分泌常压室温等离子体诱变全基因组重测序
i>Pichia pastoris</idroplet microfluidicsprotein secretionatmospheric pressure room temperature plasma mutagenesiswhole genome resequencing
郑梦林,杨艳坤,白仲虎.液滴微流控筛选提高毕赤酵母异源蛋白分泌量[EB/OL].(2024-02-06)[2025-08-18].http://www.paper.edu.cn/releasepaper/content/202402-56.点此复制
评论